Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.     

Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.     

Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry

Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.     

Integration of mass spectrometry in analytical biotechnology.

Mass spectrometry (MS) has become an indispensable tool for peptide and protein structure analysis because of three unique capabilities that enable it to be used to solve structural problems not easily handled by conventional techniques. First, MS is able to provide accurate molecular weight information on low-picomole amounts of peptides and proteins independent of covalent modifications that may be present. Second, this information is obtainable for peptides present in complex mixtures such as those that result from a proteolytic digest of a protein. Third, by using tandem MS, partial to complete sequence information may be obtained for peptides containing up to 25 amino acid residues, even if the peptides are present in mixtures. Sensitivity and speed of the MS-based approaches now equal (and in some cases exceed) that of Edman-based sequence analysis. In this perspective we discuss how MS, tandem high-performance MS, and on-line liquid chromatography/MS using fast atom bombardment or electrospray ionization have been integrated with more conventional techniques in order to increase the accuracy and speed of peptide and protein structure characterization. The expanding role of matrix-assisted laser desorption MS in protein analysis is also described. The unique niche that MS occupies for locating and structurally characterizing posttranslational modifications of proteins is emphasized. Examples chosen from the authors' laboratory illustrate how MS is used to sequence blocked proteins, define N- and C-terminal sequence heterogeneity, locate and correct errors in DNA- and cDNA-deduced protein sequences, identify sites of deamidation, isoaspartyl formation, phosphorylation, oxidation, disulfide bond formation, and glycosylation, and define the structural class of carbohydrate at specific attachment sites in glycoproteins.     

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.     

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.     

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.     

Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads

Immobilized metal ion affinity chromatography (IMAC) is a useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometry is a very suitable method for exact molecular weight determination of IMAC-isolated phosphopeptides, due to its inherent high sensitivity. Even exact molecular weight determination, however, is not sufficient for identification of the phosphorylation site if more than one potential phosphorylation site is present on a peptide. The previous method of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removal prior to MS analysis of the peptides, which can lead to sample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencing of IMAC-enriched peptides has been performed on IMAC beads applied directly to the MALDI target. The utility of this new method has been demonstrated on a protein with unknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beads resulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation site determination is unambiguous, even with a peptide containing four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beads does not adversely affect the high-mass accuracy of an orthogonal injection QqTOF mass spectrometer, making it a suitable technique for phosphoproteomics.     

Ion mobility separation of isomeric phosphopeptides from a protein with variant modification of adjacent residues

Ion mobility spectrometry (IMS), and particularly differential or field asymmetric waveform IMS (FAIMS), was recently shown capable of separating peptides with variant localization of post-translational modifications. However, that work was limited to a model peptide with Ser phosphorylation on fairly distant alternative sites. Here, we demonstrate that FAIMS (coupled to electrospray/mass spectrometry (ESI/MS)) can broadly baseline-resolve variant phosphopeptides from a biologically modified human protein, including those involving phosphorylation of different residues and adjacent sites that challenge existing tandem mass spectrometry (MS/MS) methods most. Singly and doubly phosphorylated variants can be resolved equally well and identified without dissociation, based on accurate separation properties. The spectra change little over a range of infusion solvent pH; hence, the present approach should be viable in conjunction with chromatographic separations using mobile phase gradients.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

SALSA: a pattern recognition algorithm to detect electrophile-adducted peptides by automated evaluation of CID spectra in LC-MS-MS analyses

A pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) has been developed to rapidly screen large numbers of peptide MS-MS spectra for fragmentation characteristics indicative of specific peptide modifications. The algorithm facilitates sensitive and specific detection of modified peptides at low abundance in an enzymatic protein digest. SALSA can simultaneously score multiple user-specified search criteria, including product ions, neutral losses, charged losses, and ion pairs that are diagnostic of specific peptide modifications. Application of SALSA to the detection of peptide adducts of the electrophiles dehydromonocrotaline, benzoquinone, and iodoacetic acid permitted their detection in a complex tryptic peptide digest mixture. SALSA provides superior detection of adducted peptides compared to conventional tandem MS precursor ion or neutral loss scans.     

Mining a tandem mass spectrometry database to determine the trends and global factors influencing peptide fragmentation

A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.     

Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition

A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.     

Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

Identifying proteins and their modification states and with known levels of confidence remains as a significant challenge for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and amino acid residue sequencing using high-accuracy MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3 132 confident peptide identifications ( approximately 5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3 359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed approximately 5% of the 3 359 identifications to be incorrect and many more as potentially ambiguous (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including nontryptic peptides, peptides with a variety of expected/unexpected chemical modifications, known/unknown post-translational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

High-throughput identification of proteins and unanticipated sequence modifications using a mass-based alignment algorithm for MS/MS de novo sequencing results

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.